The Role of Cyclosporin A, Leptin, and FK-506 in Leishmania
major Infections in Mice
For the Degree
Master of Arts
By
Shannon M. Potter
Under the Direction of
James K. Mitchell, PhD
Ball State University
Muncie, Indiana
May 2009
Shannon M. Potter
Table of Contents
Chapter 1: Abstract
Chapter 2: Introduction
Chapter 3: Literature Review
Leishmania
Figure 1
The Immune Response
Figure 2
Immune Response to Leishmania
Cyclosporin A
FK506
Figure 3
Leptin
Figure 4
Chapter 4: Materials and Methods
Mice
Leishmania Infection and Maintenance
Cyclosporin A
Leptin
FK506
Control Mice
Limiting Dilution Assay
Flow Cytometry Analysis
Chapter 5: Results
Table 1
Figure 5
Figure 6
Figure 7
Figure 8
Figure 9
Figure 10
Chapter 6: Discussion
Chapter 7: References
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Shannon M. Potter
Chapter 1: Abstract
The immune response to some pathogenic microorganisms fails to protect the individual
from severe infection and disease. Subsets of lymphocytes play a role in the outcome of
an infection, particularly two subsets of T cells, called T-helper ( TH1 and TH2)
lymphocytes. When preferentially stimulated, the TH2 cells are often inadequate or
inappropriate in controlling certain microbes and as a result serious infection develops.
The TH1 response, on the other hand, may result in the resolution of the severe infection.
In this study, we attempted to determine if leptin, cyclosporin A (CsA), and/or FK506
could switch the immune response in Leishmania major infected BALB/c mice from a
TH2 to a TH1 type response thereby protecting them from severe disease. Groups of mice
were infected with L. major and treated prophylactically with one of the above three
drugs. The mice were compared to non-treated, infected animals using a variety of
observations including ulcer development, footpad size, quantification of L. major in
tissues and cytokine production. Data suggests that FK506 and CsA are very effective
immunomodulatory drugs, while the amount of leptin is critical for the balance between
TH1 and TH2.
Shannon M. Potter
Chapter 2: Introduction
Leishmania spp. are parasitic protozoa that cause a spectrum of mild to severe cutaneous
diseases, as well as systemic disease in which severe enlargement of the spleen, liver, and
lymph nodes occurs. There are about 2 million new cases of leishmaniasis each year and
a tenth of the world’s population is at risk for this infection (1). This organism is found in
Africa, the Middle East, South and Central America, and India. The protozoan is
transmitted by the sand fly, a biting insect belonging to the Phlebotamine or Lutzomyia
genera. Whereas Leishmania major causes cutaneous ulcers in humans, it causes a
systemic disease in some strains of inbred mice and is similar to the severe disease
caused by L. donovani in humans. Although leishmaniasis is an extremely prevalent and
devastating disease among many people throughout the world, our ability to control this
disease through drugs or pesticides has proven inadequate. Furthermore our
understanding of the pathology of these diseases is limited, leaving many unanswered
questions including mysteries about the immune response to leishamaniasis.
The immune response to some pathogenic microorganisms, like Leishmania spp.,
sometimes fails to protect the individual or an entire strain of animals from severe
infection and disease. Although an immune response is mounted, it is not protective or
curative, it becomes destructive, causing a systemic infection. When preferentially
Shannon M. Potter
stimulated, the TH2 cells are often inadequate or inappropriate and result in a failure to
control certain microbial infections. The TH1 response, on the other hand, may result in
the resolution of the infection. The balance between these two subsets often determines if
an infection progresses or resolves itself. This balance is controlled by cytokines secreted
by the two different subsets of lymphocytes and other cells in the body. Cytokines
interact with cellular surface receptors and direct the cells in what to secrete, produce, or
stop producing. Essentially, the TH1 subset is stimulated by cytokines produced and
secreted by other TH1 cells and inhibited by cytokines produced and secreted by TH2
cells. The opposite is true for the stimulation and inhibition of the TH2 subset.
An inbred strain of mice, called BALB/c, is very susceptible to Leishmania
major. It is believed that the resulting severe illness is due to the type of response these
mice mount against the pathogen. It has been shown that these mice mount a TH2 type
response resulting in the production of antibodies and B cells that are ineffective against
the intracellular parasite. It is believed that if this response can be suppressed or reversed,
and if a TH1 type response is stimulated, increasing cell-mediated immunity, these
animals would not succumb to the illness. We attempted to accomplish this by treating
the mice prophylactically with cyclosporin A (CsA), leptin, or FK506. CsA and FK506
are commonly regarded as immunosuppressents and leptin, a naturally occurring
hormone, is an appetite suppressant with immunomodulatory properties. Preliminary
work in our laboratory has suggested that these treatments alter the susceptibility of these
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4
mice and makes them more resistant to sever disease following infection with L. major
(2).
Shannon M. Potter
Chapter 3: Literature Review
Leishmania
Leishmania species are obligate intracellular parasites that are found throughout
the world. Different Leishmania species cause a variety of infections in humans including
the frequently fatal visceral leishamaniasis or Kala Azar, self healing tropical sores or
disfiguring mucocutaneous leishmaniasis. Visceral leishmaniasis in humans is
characterized by fever, weight loss, hepatosplenomegaly, and anemia and is caused by L.
donovoni. It is often fatal even following treatment and is persistently fatal without
treatment (1). Mild cutaneous leishmaniasis is characterized by a skin ulcer at the site of
the sandfly bite. It is usually self-healing within three to six months. A rare form of this
disease, called diffuse cutaneous leishmaniasis, presents as disseminated skin lesions. An
uncommon consequence of cutaneous leishmaniasis, which can present itself years after
the initial skin ulcer heals. It is a metastatic complication of the primary lesion resulting
in disfiguring and progressive ulceration at the nasal mucocutaneous junction (1). This is
called mucosal leishmaniasis.
These diseases are extremely prevalent in Africa, the Middle East, parts of
Europe, Asia, and in Central and South America. They are transmitted by the sand fly, a
biting insect belonging to the Phleobotamine or Lutzomyia genera. Although Leishmania
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6
major causes a cutaneus form of leishmaniasis in humans, in the mouse it serves as an
animal model for the severe systemic form of the disease.
The sandfly’s natural hosts are rodents, small mammals, and dogs; humans are
accidental hosts (1). Upon biting an infected host, the sandfly ingests the amastigote
form of the parasite. The amastigotes migrate to the hindgut and transform into
promastigotes, which undergo asexual reproduction (3). The promastigotes are the
infective form of the parasite. The promastigotes then migrate forward to the esophagus
of the sandfly, clogging the esophagus. They are inoculated into the victim’s blood
stream when the sandfly bites and forces the blockage from its system. The promastigotes
are ingested by the victim’s macrophages and undergo another transformation back into
amastigotes. The amastigotes asexually reproduce within the macrophage and cause the
infected macrophage to rupture. The released amastigotes are engulfed by other
macrophages and the cycle repeats itself (3).
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7
Figure 1: The life cycle of Leshmania spp. The sandfly injects the promastigote form of
the parasite into the skin. Macrophages ingest the promastigotes. The promastigotes
transform into amastigotes and multiply in the body’s cells. The sandfly ingests
macrophages infected with amastigotes, the amastigotes transform into the promastigote
stage in the sandfly’s midgut where they multiply. The promastigotes then migrate to the
proboscis and the cycle repeats (47).
L. major uses the host complement receptors to gain access to the hostile
environment of the phagolysosome. Activated macrophages and T-lymphocytes are
recruited to the site of infection (4). Once inside the phagolysosome, L. major utilizes the
host-cell’s cyclophilin proteins as part of their replication mechanism (5). Apparently, in
humans and animals, the outcome of the disease is correlated with the type of T-helper
response. Resistance is associated with the TH1 response; susceptibility is associated with
the TH2 response. The TH1 lymphocytes primarily produce INF- and IL-2 and the TH2
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8
lymphocytes primarily produce IL-4 and IL-10. The TH1 response induces a cell
mediated (CMI) response whereas the TH2 response induces humoral immunity. CMI is
the more effective response toward Leishmania because it is an obligate intracellular
parasite in animals. It appears that a humoral response has little or no effect on the
parasite itself because the antibodies produced cannot bind to the intracellular parasite.
Indeed, even in the presence of high antibody levels, severe systemic disease can
develop. The depressed TH1 response and the activated TH2 response is an important
factor in the visceral leishmaniasis disease process. This apparently leads to
hyperglobulinema, the presence of excessive amounts of antibodies in the blood stream, a
hallmark characteristic and diagnostic of visceral leishmaniasis (3).
Visceral leishmaniasis has become increasingly prevalent with the spread of HIV
(1). With the onset of AIDS, previously “healed” or asymptomatic systemic Leishmania
infections reactivate. Species that would normally cause only the cutaneous form of the
disease may present with the visceral form. Co-infections of Leishmania and HIV are
often resistant to treatment and substantially accelerate the progression of AIDS (1).
Diagnosis of cutaneous leishmaniasis is accomplished by taking scrapings from
the edge of an ulcer, staining them with Wright’s or Giemsa’s stain and observing the
smear microscopically for the parasite in the endothelial cells and the monocytes (3).
The parasites usually cannot be found in circulating blood. Cultures are also made in
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9
case the amastigotes go undetected in the smear from the scrapings (3). Treatment is
usually unnecessary because this form of the disease is normally self-healing.
Diagnosis of visceral leishmaniasis is accomplished by several techniques.
Microscopic examination of spleen, liver and other visceral tissues for amastigotes is a
primary technique (3). Although the production of antibodies by infected individuals does
not help cure the disease, it is useful in the diagnosis of the disease. Individuals have a
high titer of IgM, IgG as well as IgE antibodies. Because of the presence of these
antibodies, ELISA and indirect fluorescet antibody techniques may be used as a
diagnostic technique. Treatment of visceral leishmaniasis is often sodium stibogluconate
or pentamidine isthionate (3). Amphotericin B/lipid formulation has been shown to be an
effective treatment, however it is expensive. Miltefosine, a more affordable treatment,
and is being used more commonly, however it is not as effective as the more expensive
treatments (3). These treatments are not only expensive, but they must be administered
for several weeks and often have serious toxic side effects (3).
The Immune Response
Immunity to pathogenic microorganisms is characterized by both innate resistance
and acquired immune responses. (6). Innate immunity is not inducible, is always present,
and is an important but general defense system against pathogens. It includes physical
barriers like skin, hair, and mucosa, and chemical barriers like lysozymes, and stomach
acids. Innate immunity is not directed against a specific foreign agent (e.g. pathogen).
Acquired immunity, however, is gained through immune responses to a specific foreign
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10
substance or antigen. It is induced and stimulated by these antigens or immunogens, it
can change over time and is the basis for vaccinations. Acquired immune responses are
induced and directed against a specific foreign agent. Essentially acquired immunity has
the ability to distinguish self from non-self, and is characterized by its inducibility
specificity, memory, and regulation (6).
Acquired immunity is further broken down into two branches, humoral immunity
and cell mediated immunity (CMI). Humoral immunity is primarily mediated by
antibodies present in the plasma, lymph and tissue fluids (7). It is particularly protective
against extracellular pathogens and toxic macromolecules. There are primarily three
types of cells involved in the immune response: antigen presenting cells, T helper
lymphocytes and B cells which actually secrete the antibody. B cells synthesize and
display membrane-bound immunoglobulin, or antibody (Ab) molecules which serve as
receptors for antigens. Following stimulation with antigen and T cell lymphokines, B
cells differentiate into plasma cells which secrete antibodies, but do not express them on
their membranes. Together these cells, and molecules make up the humoral immune
response. B cells as well as dendritic cells and macrophages also serve as antigen
presenting cells (APC), which phagocytize antigens, break them down and present the
antigen to T cells on major histocompatibility complex molecules (MHC). T cells can
only recognize antigen after it is processed and displayed on an MHC class I or II
molecule.
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MHC molecules interact with T cells, via the T cell receptor (TCR). The TCR is
the T cell’s membrane bound antigen receptor. Unlike the B cell’s membrane bound
receptor, the TCR cannot recognize free antigen. Instead it recognizes antigen bound to
MHC molecules. There are two major types of mature T cells, characterized by
expression of either CD4+ and CD8+ molecules on their cell surfaces (7). CD4 and CD8
are cell surface markers/receptors.
Functionally these cells behave differently. CD8+ T cells bind with antigens
presented on MHC class I molecules (7). These cells generally function as cytotoxic T
cells (TC cells) killing virally infected cells. These cells generally recognize endogenous
peptide antigens. CD4+ T cells bind with antigens presented on MHC class II molecules.
These cells generally function as T-helper cells (TH cells) helping either other T cells or B
cells become effecter cells. There are two types of TH cells, TH1 and TH2 cells
differentiated by the lymphokines they secrete. TH1 clones are involved in cell mediated
immunity and TH2 clones are predominantly involved in humoral immunity, providing
cognate B cell help (7). The two T-helper subsets are controlled by many different
cytokines. The regulation of these subsets is shown in Figure 2 (7). The subsets
themselves produce very specific cytokines which, in turn, regulate a specific set of
immune cells directing the immune response toward humoral or cell-mediated.
The balance between TH1 and TH2 often determines the outcome of an infection.
In many chronic illnesses, a polarized TH1 or TH2 type response occurs, which is
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12
mutually exclusive response, conceivably because of the counter-inhibitory effects of
cytokines on the reciprocal TH subsets (8). Murine TH1 cells produce IL-2 and INF- but
little IL-4 and IL-5 and have been shown to play an important role in CMI responses
against intracellular pathogens (9). TH2 cells, on the other hand, produce predominantly
IL-4, IL-5, and IL-10 but little INF- and are involved in humoral immunity and allergic
responses (9).
Figure 2: Regulation of
TH1 and TH2 subsets by cytokines. Each subset of T-cell is regulated by a particular set
of cytokines. In turn, each subset produces its own set of cytokines that regulate immune
cells and direct the type of immune response that occurs. (7)
Respiratory syncytial virus (RSV) causes most cases of viral bronchiolitis, which
is the single most common cause of hospitalization of infants and young children (10). In
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the 1960s children were vaccinated with a formalin inactivated virus preparation.
Unfortunately, this vaccination enhanced the disease process when the child was exposed
to the virulent virus. The killed virus induced a humoral type response instead of
inducing a CMI type response. Therefore, the memory acquired by the immune system in
these vaccinations was a humoral type immunity, which is directed by the TH2 subset.
This apparently led to an exacerbated inflammatory response when an RSV infection
occurred. Resolution of the infection is associated with CD4+ and CD8+ T cells that
produce INF-. In these resolving infections, only low levels of IL-2, IL-4 and IL-5 were
detected which suggests a TH1 type immune response. Interestingly, in vitro studies show
the co-production of IL-4 and INF-, suggesting that the immune response is not always
completely polarized (10). Field et. al claim that the only evidence for the presence of a
pure TH1 or TH2 type response is based on clonal proliferation of cells obtained from the
peripheral blood of patients (11). In most situations, a mixture of responses exists with
shifts in either direction.
The outcome of an HIV infection also seems to be dependent on the type of TH
response. The presence of specific TH1 type responses in individuals has been shown in
persons who have been exposed to HIV but are not seropositive for HIV (12). This
suggests the presence of HIV-specific protective immunity in these individuals. Persons
who live in areas where infectious diseases are endemic seem to have a highly activated
immune system. Their peripheral blood mononuclear cells were found to secrete
significantly higher amounts of IL-2, IL-4, IL-10, and soluble TNF receptors and lower
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14
amounts of IL-6 in comparison with healthy non-Ethiopian controls (12). The people of
these countries (Africa, the middle East, parts of Europe, Asia, and Central and South
America) are often exposed to helminthic infections early in life (3). It is believed that the
helmith infections alter the balance between TH1 and TH2 in such a way that makes the
host more receptive to HIV and more vulnerable to its effects (12). The same is true for
mycobacteria infections and other intracellular parasitic infections, like leishmaniasis
(13). However, Maizels et. al. (14) show that helminthic infections steer the immune
response of the host in the direction of TH2, which is more protective for the host and
down regulates the TH1 response. Since helminthes are endemic to areas of the world that
are also endemic to Leshmania spp., Maizels et. al.’s (14) determination could explain
why some people are immune to leshmaniasis and others become seriously ill.
Immune Response to Leishmania
Murine models have been very useful in understanding the visceral disease
process. Mice resistant to L. major develop a protective immunity by the expansion of
CD4+ TH1 T cells. These protective TH1 cells result from their production of INF-,
which induces the synthesis of nitric-oxide synthase, (iNOS) (4). Nitric oxide synthase
leads to the L-arginine-dependent production of reactive nitrogen radicals, which are
toxic to the intracellular Leishmania parasite. In vitro, this cytotoxicity is dependent on a
functional Fas-FasL pathway (4). Stenger et. al. have shown that the cutaneous lesion and
draining lymph nodes of infected, clinically resistant C57BL/6 mice have a detectable
amount of iNOS earlier during infection and in significantly higher amounts than in
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infected, susceptible BALB/c mice (15). Cells isolated from lesions in infected BALB/c
mice were found to stain positively for transforming growth factor beta (TGF-), a
powerful inhibitor of iNOS in vitro (15). Susceptible mice develop an early expansion of
TH2 T-cells. These cells produce IL-4, which appears to be the major inducer for TH2 and
inhibitor for TH1 type responses (16). Treatment of BALB/c mice with anti-CD4 induced
resistant to the disease, and restored the expression of iNOS in the tussue (15). This
response is thought to be due to a parasite antigen that is a Leishmania homolog of
receptors for activated C kinase, or the LACK antigen. Mice that have been
transgenically made to express the LACK antigen in the thymus exhibit a diminished TH2
response and a healing phenotype (16). The type of response that dominates the outcome
of the infection occurs very early after detection of antigen, like Leishmania’s LACK
angtigen. CD4+ cells specific for the L. major LACK antigen were shown to expand 100fold; 70% of those cells were detected to as producers of IL-4 by hour 96 (17). BALB/c
mice have been specifically shown to produce a burst of IL-4 within the first day of L.
major infection (18). This early IL-4 production was shown to be a result of the cognate
recognition of a single epitope in a distinctive antigen, the LACK antigen (18). Schilling
et. al. (19) have demonstrated that LACK-specific T-cells are active in both the draining
lymph node and the site of infection, further promoting the rapid replication of the
parasite and its dissemination to the internal organs.
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The induction of TH1 dominated or TH2 dominated responses involves a very
complex interaction between antigen presenting cells, specific antigen epitopes (such as
the LACK antigen) and T cells. In addition to antigen TCR processing recognition, these
interactions involve a number of cell surface costimulatory molecules such as CD40,
CD28, B7molecules and ICOS. Inducer costimulator proteins (ICOS) found on the
surface of T cells have an important role in T cell induction, stimulation differentiation,
as well as Ig class switching (20). ICOS is induced by a homolog of CD28 and reacts
with B7 homolog. It enhances T cell cytokine production, up-regulates the production of
the CD40 ligand and induces Ig production by B cells. ICOS is induced upon T cell
receptor engagement with the antigen (20). Whereas another T cell co-stimulator
molecule, CD28 is constitutively expressed on most T cells (7). Expression of ICOS is
enhanced by CD28 costimulation. The inducible expression of ICOS suggests that ICOS
regulates recently activated effecter T cells. Functional studies have suggested that ICOS
is important for the TH2 response, preferentially inducing IL-4 and IL-10 production. In
contrast, other studies suggest that ICOS may also regulate the TH1 responses. Greenwald
et. al. (20) demonstrated that ICOS-/- mice infected with Leishmania mexicana exhibited
pronounced deficits in Ig class switching and T cell cytokine production. Development of
cutaneous lesions and inflammatory infiltrates was impaired in these mice. This
demonstrated that ICOS is probably a critical regulator of both the TH1 and TH2 type
responses in leishmaniasis. It is clear that much more needs to be understood about the
pathology and immune response to leishmanial infections.
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Cyclosporin A
Cyclosporin A (CsA) is a cyclic polypeptide metabolite commercially synthesized
by the fungus Tolypocladium inflatum with a molecular weight of about 1200 which has
been shown to have immunosuppressive effects (21). CsA is a lipophilic compound that
contains neither free amino nor carboxylic groups. It has a formula of C62H111N11O12 and
is a cyclic undecapeptide composed of ten amino acids and the unique amino acid Nmethyl-2-amino-3-idroxy-4-methyl-octa-6 enoic acie (MeBmt). With the exception of
one D-alanine, all of the amino acids are L-series amino acids. Seven of the amino acids
have an N-methylation, which is responsible for its lipophilic nature. Stabilization of the
CsA molecule is maintained by several hydrogen bonds. CsA is synthesized in a nonribosomal biosynthesis pathway (21). A non-ribosomal pathway is directed by a
multienzyme tiotemplate and without the direct use of nucleic acids (22). Biosynthesis
using a non-ribosomal pathway is accomplished by the formation of linear peptidyl
intermediates which may be enzymatically modified (22). It is thought that CsA is
biosynthesized in a three step process: i. Synthesis of all building units, ii. Activation of
the 11 amino acids, and iii. N-methylation and peptide formation (21).
Toxicity studies show that the selectivity of CsA is for lymphocytes and that there
is a lack of effect on haemtopoiesis (21). CsA acts selectively on lymphocytes, mainly T
cells, affecting particularly the inductive phase of immunity, rather than the proliferative
phase of immunity. CsA has been shown to alter the balance between T helper
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lymphocytes and suppressor T-cells. Its immunomodulatroy effects appear to result from
the inhibition of the production of T-cell derived soluble mediators like IL-2 and IL-3 and
INF- which leads to an inhibition of primary TH cell activation and blocks the formation
of lymphokines like IL-2. It also decreases the secretion of INF-. The production of IL-
and INF-, however does not seem to be affected (21). T-cell proliferation is inhibited
by the blockage of Ca2+ dependent pathways required for the induction of IL-2
transcription (23). Complexes of CsA inhibit the calmodulin dependent protein
phosphatase, calcineurin (24). The calcineurin pathway dephosphorylates the inactive
cytosolic form of the T cell specific nuclear factor NF-AT, which is a transcription factor
necessary for T cell activation, See Figure 3 (7).
CsA is most commonly used as an immunosuppressant for transplant patients.
Previous work in our lab has shown that in addition BALB/c mice treated
prophylactically with CsA have significantly enhanced resistance to L. major infections
(2).
FK506
FK506 is a macrolide that has a molecular weight of about 850 Daltons and is
produced by a strain of the bacterium Streptomyces (25). It has a molecular formula of
C44H49NO12H2O. FK506 has been shown to suppress the immune response both in vivo
in mice and humans and in vitro it is more potent/mg than that of CsA, and has been
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shown to suppress the immune system in vitro at about 100 times lower concentration
than CsA. It also appears to be less toxic than CsA (26).
FK506, like CsA, has been shown to strongly suppress the humoral immune
response, possibly by inhibiting the production of T-cell derived soluble mediators (26).
FK506 especially affects mature CD4+ T cell reconstitution in the periphery, seemingly
by interfering with thymic activation signals such as CD69, which is required for final
maturation of T cells (27). Both compounds have also been shown to semi-selectively
spare CD8+ T cells (25). These T cells are thought to produce cytokines that act on other
lymphocytes; for example, these cells produce INF-, which stimulates the TH1 response
(26).
FK506 binds to an immunophilin named FKBP-12, or FK506 binding protein
number 12 (28). Immunophilins serve as receptors to immunosuppressive or
immunomodulatory drugs. FK506 has also been shown to target the calcineurin pathway
in a similar manner as Cyclosporin A (Figure 3) (24, 29). The FK506/FKBP-12 complex
targets the calcium dependent protein calcineurin. This complex inhibits phosphorylation
of calcineurin. This inhibition increases the phosphorylation of NOS, a substrate for
calcineurin; calcineurin dephosphorylates the NOS protein. The phosphorylated
calcineurin enzyme diminishes its catalystic activity, suppressing the formation of NO2,
which mediates glutamate neurotoxicity. Tao and Aldskogius (28) have shown that
FK506 enhances the survival of damaged nerves by the same pathway . Nitric oxide
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(NO) inhibition prevents the glutamate-elicited neurotoxicity. FK506 may exert survivalpromoting effects by decreasing NO synthesis via the inhibition of NOS, which is
dependent of calcium for its activation (28). Down-regulation of NO, in turn, inhibits
glutamate release and protects against neurotoxicity.
Figure 3: The Calcineurin Pathway and the Role of CsA and FK506: Calcineurin
dephosphorylates inactive NFAT, which activates the transcription of cytokine genes.
CsA and FK506 have been shown to block this pathway by inhibing the
dephosphorylation of Inactive NFAT. This, in turn, inhibits the transcription of cytokine
genes (5).
Calcineurin also dephosphorylates the cytoplasmic nuclear factor of activated T
cells, called NF-ATc (30). This induces its nuclear translocation and eventually forms a
functional transcription factor that activates the transcription of IL-2. Therefore, FK506
also inhibits the synthesis and subsequent secretion of IL-2, which is the cytokine that
induces the clonal proliferation of T cells by suppressing calcineurin’s phosphatase
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21
activity. Calcineurin has been shown to co-localize with FKBP-12 in neurons (31). This
co-localization in the nerve, along with the inhibition of calcineurin by FK506 is believed
to result in the promotion of neuroregeneration by enhancement of growth-associated
protein-43 (GAP-43) (32). GAP-43 is a phosphoprotein that has a key role in nerve
growth, which facilitates neuronal growth after injury.
Agostinho and Oliveira (33) studied the effect that FK506 and CsA has on
diseases that are caused by prions in the brain. Prions are proteins that are enriched with
-sheet conformations, which makes them resistant to proteolytic cleavage by proteases.
Alzheimer’s disease, the most common form of dementia in the elderly, has been shown
to be associated with the amyloid beta protein (A) (34). The A protein results from the
abnormal proteolytic cleavage of amyloid precursor protein. Agostinho and Oliveira (33)
also studied prio-related encephalopathies (PRE), which are caused by the scrapie
isoform of prion protein (PrPSc). Plaques formed by the presence of prions consist of a
core surrounded by degenerated neuritis, microglia and astorcytic processes. The
apoptotic pathway of neuronal death appears to be a prominent feature of both
Alzheimer’s disease and PRE. Apoptosis is thought to be regulated by the calcineurin
pathway through certain cystine aspartate-specific proteases (caspases). Caspase-3 is
thought to be a major player in this apoptotic pathway. Agostinho and Oliveira (33) used
FK506 to determine if the calcineurin pathway is involved in neuronal death induced by
the A prion and the PrPSc prion. They found that FK506 prevented the release of cyt c
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and thereby prevented the activation of caspase-3. This indicated that this phosphatase is
involved in the neurotoxic mechanism induced by A and PrPSc. These studies also
indicated that FK506 did prevent neuronal cell death.
FK506 is currently being used as an immunosuppressant in transplant patients. It
has been shown to reduce graft vs. host disease or rejection, which CsA does not (27).
Previous work in this laboratory has shown that FK506 also significantly enhances the
resistance to Leishmania major infections in BALB/c mice (unpublished data).
Leptin
Leptin is a recently discovered hormone that is secreted exclusively by
adipocytes. It is a 16-kDa nonglycosylate peptide hormone (35). The primary amino acid
sequence of leptin indicates that it may adopt a helical cytokine structure similar to IL-2
(36). The leptin receptor is expressed not only within the nervous system, but also within
the immune, hematopoietic, and reproductive systems (37). Leptin induces proliferation,
differentiation, and functional activation of hemopoietic cells (38). The leptin receptor
has a sequence homology to the members of the cytokine receptor superfamily (39). It
also increases the expression of the early activation marker CD69 in monocytes but not in
lymphocytes. Cytokine production is enhanced, and the basal expression of activation
markers such as CD38 in human monocytes are stimulated as previously described in the
LPS stimulation system (33). Leptin, when given to lean or obese mice, increases basal
metabolic rate and reduces food intake, leading to weight loss.
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Leptin has been shown to be essential in balancing the susceptibility to infection
and autoimmunity, as shown in Figure 4 (39). Normal leptin levels balance the TH1 and
TH2 response, whereas if leptin levels fall too low, TH1 type responses are reduced and
TH2 type responses are increased. If too much leptin is present there is a reduced TH1
type response and an altered inflammatory response. Circulating levels of leptin are
proportional to fat mass, but may be lowered rapidly by fasting or increased by an
inflammatory response (39). An impaired cell mediated type response, or a TH1 type
response, and reduced leptin levels are both features of low body weight. Starvation or
malnutrition, which decreases leptin levels, results in the suppression of TH1 mediated
inflammatory responses (40). It is well established that nutritional deficiency impairs cell
mediated immunity, phagocyte function, and cytokine and antibody production (41).
Both naturally leptin-deficient mice and leptin-receptor-deficient mice, despite
their great body-fat mass, have thymic and lymph node atrophy, reduced numbers of
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24
Figure 4: Leptin’s effect on TH1 vs. TH2 balance. Normal levels of plasma lepitn
promote a balance between TH1 and TH2 type responses. Too much leptin or too little
leptin cause this balance to shift (39).
bone-marrow precursors, and impaired cell mediated immunity (39). In rodents, it has
been shown that after immunization females have a more vigorous T cell and antibody
response than males. The presence of estrogens produce higher levels of TH1 cytokines
and they show a consistent in vitro secretion of INF- and IL-1 (42). Androgens and
testosterone produced by males increase the production of IL-4 and IL-5 and switch to
the TH2 type response (42). Serum levels of leptin are sexually dimorphic, being higher in
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25
females than in males (42). This suggests that leptin is involved in gender-related
differences in susceptibility.
Human leptin has been shown to enhance cytokne production by murine
peritoneal macrophages. Santos-Alvaraz et. al. (37) have demonstrated that human leptin
stimulates proliferation in a dose-dependent manner and functionally activates human
circulating monocytes in vitro by inducing the production of cytokines such as TNF-
and IL-6. The expression of different activation markers in human monocytes, like CD25
(IL-2 receptor), CD71 (transferring receptor), and CD38 were also increased by leptin in
a dose-dependent manor. The expression of other activation markers that were already
present at high levels on the surface of resting monocytes, (e.g. HLA-DR, CD11b, and
CD11c), were also enhanced by leptin in a dose-dependent manor (37). Santos-Alvaraz
et. al. (37) also demonstrated that the stimulation of PBMC with leptin produced an
increase in the percentage of monocytes producing cytokines. The effect of leptin was
more evident on the population of monocytes producing IL-6, from which about 50% coexpressed TNF-. However, the population of monocytes producing only TNF- was
less affected. Therefore a greater effect of leptin on monocyte IL-6 synthesis might be
expected. The pro-inflammatory response of these cytokines may be enhanced by leptin,
functioning as an amplification signal for the activation of monocytes. Monocytes are
activated by leptin through the induction of proliferation expression of activation
markers, and the production of cytokines.
Shannon M. Potter
26
Martín-Romero et. al. (38) have shown that leptin modulates CD4+ T lymphocyte
activation toward the TH1 phenotype by stimulating the synthesis of IL-2 and INF-.
Leptin receptors are expressed on both CD4+ and CD8+ cells, as well as hemopopoetic
cells, other lymphocytes, and adpocytes. The leptin receptor has a sequence homology to
members of the cytokine receptor class I superfamily (44). The receptor has been shown
to have similar signaling capabilities to that of the IL-6 type cytokine receptor (45).
Martin-Romero et. al. (38) attempted to determine the effects of human letpin on
activation, proliferation, and cytokine synthesis in peripheral human lymphocytes. They
found that a leptin dose-dependent enhances the proliferation and activation of circulating
T-cells when they were costimulated with either PHA or ConA. Alone, leptin was unable
to activate T-cells. Leptin also dose-dependently enhanced stimulated CD69 expression;
late activation markers CD25 and CD71 were also activated by leptin stimulation. Leptin
modulated CD4+ T-cell activation toward the TH1 phenotype by stimulating the
production of IL-2 and INF-. This shift toward the TH1 phenotype may have some
relevance in the pathophysiology of immunologic alterations related to obesity. Upon
measuring the levels of IL-2, INF-, and IL-4, they found that IL-2 and INF- levels were
increased and that IL-4 was undetectable under the experimental conditions investigated.
This suggests that leptin levels in the blood stream can easily shift the TH1/TH2 balance.
According to Lord et. al. (46), “Leptin increases the TH1 and suppresses the TH2
cytokine production. Without leptin present, the TH2 response is favored; leptin seems to
Shannon M. Potter
27
induce the switch to the TH1 response”. Leptin modulates the production of IL-2 and
INF-, which are TH1 activators (38). Circulating levels of leptin have been found to be
proportional to fat mass (43). These levels may be lowered by rapidly fasting or
increased by inflammatory mediators (47, 48). An impaired cell mediated response and
reduced levels of leptin have both been found to be features of low body weight in
humans (49, 50).
Lord et. al. (46) found that leptin has a specific effect on T-cell responses,
differentially regulating the proliferation of naïve and memory T-cells. They also tested
the possibility of leptin influencing TH cytokine production by measuring IL-2, INF-,
and IL-4 levels in MLR experiments (46). They found that leptin increased IL-2
production and showed a substantial increase in INF- secretion. IL-4 production was
completely inhibited, suggesting that leptin may bias T-cell responses towards a TH1
phenotype and suppress the TH2 phenotype. In the absence of leptin, TH2 responses were
dominate, the addition of leptin seemed to induce a switch from a TH2 response to a TH1
response. They also found that T-cells, rather than accessory cells, were the target of
leptin action. Cognate recognition by T-cell antigen receptor was required before leptin
could take effect. This indicated that the enhancement of proliferation observed was due
to a specific effect of leptin receptor signaling rather than to a non-specific mitogenic
stimulus (46).
Shannon M. Potter
Chapter 4: Materials and Methods
Mice
BALB/c mice were originally obtained from Harlan-Sprague Dawley, Inc.
(Indianapolis, IN) and were bred in our own mouse colony (Department of Biology) .
Both male and female mice were used with ages ranging from 6 to 12 weeks. The mice
were housed in a light and temperature controlled environment with a light/dark cycles of
approximately 12 hours each.
Leishmania Infection and Maintenance
The Leishmania major (Freidlin strain, NIH) pathogen was originally provided by
Dr. D. Sacks. The pathogenicity of the Leishmania was maintained by passage through
BALB/c mice on a regular basis. To accomplish this, the mouse was infected via the left
rear footpad with about 1.0 X 106 organisms per ml. The infection was allowed to
progress for two months. These mice were used as the source of pathogenic L. major by
removing the spleen and/or popliteal lymph node (located between the knee joint and the
articular end of the femur) and culturing the cells. The intracellular amastigote form of L.
major was released from the hosts’ cells. These pathogens then developed into the
infective promastigote form when grown in vitro. The promastigotes were grown at 25
C and passed in culture approximately once every week as described below.
Shannon M. Potter
29
The media used to grow the pathogen was Dulbecco’s M199 (Sigma Chemical
Co., St. Louis MO) amended with 1M HEPES, 10 units penicillin, 10 g/L streptomycin,
0.35g/L sodium bicarbonate, and 20% fetal calf serum. The pH of the medium was
adjusted to 7.2. For all of the experiments, promastigotes, which had not been passed in
vitro more than five times, were injected into the left hind footpad with a dose of 1.0 X
106 organisms per ml. Over a series of 7 weeks, the middle of the infected footpads were
measured with a vernier caliper gauge. The regional draining lymph nodes (popliteal)
were also palpated to indicate the spread of infection. Swelling of the popliteal lymph
node was indicative of the spread of infection.
Cyclosporin A
Cyclosporin A (CsA) was originally obtained by Sandoz, Ltd., East Hanover, NJ.
It arrived prepared as an oral solution at concentration of 100mg/ml in olive oil in a
labrafil base. A group of 15 mice, about 12 weeks in ages were each given a dose of CsA
at 75 µg/g of body weight twice daily for fourteen days, beginning one day prior to
infection with L. major for both trials. The CsA was administered using a 20 l pipetmen
and a plastic tip. The CsA was squirted into the mouth of the mouse. The mice were
weighed before the administration of the drug to determine the dose and to observe the
effect of the drug on the weight of the mice. The health of the mice were observed by
noting the general appearance of the animals, texture of the fur, and the evidence of
diarrhea in the cages. The animals were observed for smooth, ruffled or oily fur.
Shannon M. Potter
30
Leptin
Murine leptin was purchased from Sigma Chemical Co., St. Louis, MO. The
leptin used was in a solution of 0.2mg/ml in sterile PBS. During Trial 1, a group of 12
mice at 8 weeks in age were treated with1.8 g/g of body weight twice daily for five
days, beginning two days prior to infection. During trial 2, a group of 15 mice at 8 weeks
in age were treated with 2.0g/g of body weight twice daily for five days, beginning two
days prior to infection. The different rates were used due to the fact that level of leptin in
the body determines the type of TH response (39). The leptin was administered with a ½
cc insulin syringe with a 30 gauge needle. The injections were given intraperitoneally.
The mice were weighed before the administration of the drug to determine the dose and
to observe the effect of the drug on the weight of the mice. The health of the mice was
observed by noting the general appearance of the animals, texture of the fur, and the
evidence of diarrhea in the cages, as described above.
FK506
Fujisawa Pharmaceuticals USA, Inc., Aurora, IL generously donated the
powdered form of FK506. The powder was reconstituted in 0.2% w/v
carboxymethylcellulose (CMC) at a concentration of 2.5 mg/ml. The CMC was prepared
in a 1% stock solution by dissolving CMC in sterile water. This solution was then diluted
to creating a working solution at 0.2% w/v. A group of seven mice at an age of 8 weeks
were used for the first trial and a group of 15 mice at an age of 8 weeks were used for the
second trial. These mice were given a dose of 50 g/g of body weight once daily for 7
Shannon M. Potter
31
days, beginning one day prior to infection. The FK506 was administered
intraperitoneally using a 1 cc syringe and a 26-gauge needle. The mice were weighed
before the administration of the drug to determine the dose and to observe the effect of
the drug on the weight of the mice. The health of the mice were observed as previously
described.
Therapeutic FK506
FK506 was also used in a small group of mice (5 mice) in a therapeutic treatment
regimen. These mice, along with a control group (infected, non-treated), were infected
with 1X106 Leishmania promastigotes/ml. Fourteen days post infection treatment with
FK506 was given at a dose of 50 g/g of body weight intraperitoneally using a 1 cc
syringe and a 26-gauge needle. Control mice were given 0.2% w/v CMC
intraperitoneally with a 1 cc syringe and a 26-gauge needle. Both groups were treated
once daily for seven days. Health was monitored as described previously.
Control Mice
A group of 20 mice at 16 weeks in age were used as a control group. These mice
were given sterile 0.2% w/v CMC intraperitoneally for seven days beginning one day
prior to infection. This dose was based on the amount of fluid that would have been
given if they were in the FK506 group. It was administered via a 1cc syringe and a 26
gauge needle. The mice were weighed before the administration of the solution to
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32
determine the dose and to observe the effect of the solution on the weight of the mice.
The health of the mice was observed as described previously.
Limiting Dilution Assay
The limiting dilution assay is based on techniques described by Buffet et al (51).
One animal from each group was sacrificed either by cervical dislocation or by CO2
asphyxiation. The spleen and popliteal lymph node was removed aseptically and placed
in a sterile petri dish containing 2 ml of complete Dulbecco’s M 199 medium. Twenty
milligrams of each organ was aseptically weighed for use in the assay. To release the L.
major, each organ was ground between two sterile frosted glass slides. The ground tissue
was then placed into 20 ml of complete Dulbecco’s M 199 medium to make a 1mg/ml
suspension. A duplicate series of 12 sterile tubes containing 2.5 ml of complete
Dulbecco’s M 199 medium amended with 20% fetal calf serum was prepared. Zero point
five milliliters of the stock suspension was placed in the first tube and then 1:6 serially
diluted. The tubes were incubated at room temperature (23˚C - 24˚C) for at least 3
weeks. The presence of any L. major promastigotes microscopically at 400x indicated a
positive result for that dilution and represented the minimal most probably number
(MPN).
Flow Cytometry Analysis
The procedure used in the flow cytometry analysis of lymphocytes extracted from
the popliteal lymph node was based on Barbara Foster and Calman Prussin’s protocols
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33
(52). Popliteal lymph nodes were removed from three animals in each being tested. The
organs were ground between sterile frosted slides and washed in RPMI medium (52).
Cells were suspended in complete, pre-warmed RPMI at a concentration of 2 X 106
cells/ml and split into polystyrene round bottomed tubes containing 2 ml each. AntiCD3, anti-CD28 and L. major antigen was added to each tube, and the tubes were
centrifuged at low speed (300xg) for five minutes to pellet the cells in close proximity
with each other, the antibodies, and the antigen stimulant. Tubes were then incubated in
the CO2 incubator at 37 C for 2.5 hours. A 200µg solution of BFA was added to each
tube to prevent the export of intracellular cytokines being produced. Cells were then
incubated for an additional 4 hours. After incubation, cells were washed with 0.1 M
EDTA and transferred to 4 ml V-bottomed tubes. The cells were fixed in pre-warmed
4% Para-formaldehyde, washed again with cold PBS and then resuspended in 10%
DMSO/PBS. Cells were counted and placed in freezer vials to be stored at –80 C until
staining could be performed.
The staining of stimulated, frozen cells was also adapted from Prussin’s protocols
(52). The cells were thawed in a 37 C water bath for 10 minutes. Pre-warmed 25µl PBSSaponin (PBS-S) was added to each sample of cells, and the cells were transferred to Vbottomed tubes. Cells were centrifuged for 10 minutes at high speed (1500xg), and the
pellet was resuspended in PBS-S/milk. The milk is used to block non-specific binding
proteins (52). The cells were then placed on ice for 30 minutes to allow the milk to act as
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34
a blocking agent. One tube from each group was chosen as a control tube, and the
contents of this tube split in-half. One-half of the cells were resuspended in unlabeled
monoclonal antibodies against IL-4 and INF-. The other half was resuspended in nonspecific rat IgG as an isotype control. The rest of the tubes were resuspended in PBSS/milk. All tubes were placed on ice for 1 hour. After chilling, labeled antibodies against
IL-4 and INF- were added to each tube, and the tubes were again placed on ice for 1
hour, in the dark. Cells were then washed in PBS-S and resuspended in PBS/BSA. Cells
were counted in flow cytometer within 24 hours.
Shannon M. Potter
Chapter 5: Results
Effects of drug toxicity on the health of the mice is shown in Table 1. The
infected, non-treated mice experienced mild weight loss, mild roughness of the coat, zero
deaths, and mild diarrhea. The mice treated with CsA, which is known for its toxicity,
experienced mild to severe weight loss, severe roughness of the coat, zero deaths, and
severe diarrhea. After the treatment period ended, the overall appearance of these
animals returned to normal. Mice treated with FK506 experienced very mild weight loss,
very mild roughness of the coat, very mild diarrhea, and zero deaths. Leptin treated mice
had symptoms that fell in-between the severity of the CsA treated mice and the mildness
of the FK506 treated mice.
The measure of footpad thickness was used an indicator used in determining
disease process. Normal, healthy mice have and average footpad thickness of about 2mm.
Figure 5 shows the average footpad thickness of mice in each treatment group and the
non-treated infected group during Trial 1. The control group (non-treated) was challenged
with L. major but not treated with any immunomodulatory drug. The mice in this group
experienced a large amount of swelling that dropped of by week 17. This drop in footpad
thickness was due the large amount of ulceration. Which caused a loss in foot tissue.
During trial 2, the dose of leptin was increased from 1.8 g/g of body weight to
2.0 g/g of body weight. In the first trial, leptin seemed to delay the disease, however, in
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36
Table 1 Drug
Toxicity
Drug Used
Observed Weight
Loss
Smoothness of Coats
by Visual Inspection
Overall Health by
Visual Inspection
Controls (0.2% CMC)
Mild
Mild Roughness
Fair, 0 Deaths, Mild
Diarrhea
CsA
Mild-Severe
Severe Roughness
Fair, 0 Deaths, Severe
Diarrhea
FK506
Very Mild
Very Mild
Fair, 0 Deaths, Mild
Diarrhea
Leptin
Mild
Mild Roughness
Fair, 0 Deaths, Mild
Diarrhea
Table 1: Drug Toxicity. CsA was the most toxic of the drugs used, mice treated with
this drug experienced severe diarrhea and mild to severe weight loss during the treatment
period. Their fur was also severely ruffled. Mice treated with FK506 developed very
mild diarrhea and very mild roughness of the fur. Leptin treated mice fell somewhere inbetween, with mild weight loss, mild diarrhea, and mild roughness during the treatment
period.
37
Shannon M. Potter
Footpad Thickness (mm)
Figure 5: Average Footpad Thickness for Trail 1
6
5.5
5
4.5
4
3.5
3
2.5
2
1.5
1
0.5
0
Controls
CsA 75mg/kg
FK506 50 ug/g
Leptin 1.8 ug/g
0 1 2 3 4 5 6 7 8 9 10 17 18
Week of Infection
Figure 5: Average Footpad Thickness of Mice During Trial 1. Mice treated with
FK506 developed very little swelling and no ulcers. Mice treated with CsA were similar
to mice treated with FK506, little swelling occurred and no ulcer development occurred.
Mice treated with leptin did experience more swelling and ulcer development. Non
treated mice developed large amounts of swelling and ulcer development. The size of the
footpads of the non-treated mice dropped due to necrosis of foot tissue.
Shannon M. Potter
38
the second trial the mice treated with leptin developed ulcers and swelling weeks before
the non-treated animals. The mice treated with CsA experienced little swelling and no
ulcer development. Mice treated with FK506 did not develop any swelling, redness, or
ulcer development (Figure 6).
To determine parasite load, MPN dilution assays were performed on the draining
regional lymph node and the spleen of animals from each group. Figure 7 presents
average titers obtained from the popiteal lymph nodes. Titers were averaged together,
data without standard error bars are an average of two animals, data with standard error
bars are an average of four animals. Animals treated with FK506 did not have very high
parasite loads. The parasite titer did drop in the FK506 animals by week 12. Animals
treated with leptin had particularly high parasite titers, which also dropped by week 12.
The parasite load in animals treated with CsA fluctuated throughout the experiment, but
never reached levels as high as leptin animals or as low as FK506 animals. Infected, nontreated animals had parasite titers that increased fairly steadily with time. Only the
spleens of animals in the infected non-treated group and the CsA group had positive
titers. These positive results only occurred during weeks 6 and 12 (data not shown).
To determine the type of immune response occurring in the FK506 and in the
infected non-treated animals, flow cytometry analysis of stained intracellular cytokines
was used. Later, we also analyzed cells stained for intracellular cytokine production from
all of the experimental groups. Figure 8 shows the percent of cells in each sample that
Shannon M. Potter
39
Figure 6: Trial 2 Average Footpad Thickness
Average Footpad Thickness
(mm)
5
4.5
4
Non-treated
3.5
CsA 75mg/kg
3
FK506 50ug/g
2.5
Leptin 2ug/g
2
1.5
1
0
1
2
3
4
5
Weeks Post Infection
6
7
Figure 6: Average Footpad Thickness of Mice during Trial 2. FK506 and CsA treated
mice did not have much swelling and did not develop ulcers. The mice in the leptin
treated group experienced a large amount of swelling and ulcer development. The ulcer
development occurred before the mice in the control, non-treated group.
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40
Figure 7: Limitng Dilution Assay of Popliteal
Lymph Nodes
Log 10 (parasites/mg of tissue)
12
10
8
Non Treated
CsA
6
Leptin
4
FK506
2
0
Week 3
Week 4
Week 6
Week 12
Weeks Post Infection
Figure 7: Limiting Dilution Assays of Popliteal Lymph Nodes. Data without error bars
are an average of 2 animals, data with error bars are an average of 4 animals.
Shannon M. Potter
41
Figure 8: Amount of Intracellular INF-g Produced
3.5
% Positive Cells
3
2.5
Non-treated
FK506
Leptin
CsA
2
1.5
1
0.5
0
Week 3
Week 4
Week 6
Weeks Post Infection
Figure 8: Amount of INF- produced by mice infected with L. major. Three weeks
post infection, mice treated with FK506 were producing less INF- than mice that were
infected but not treated. By four weeks post infection, the mice treated with FK506 were
producing slightly more INF-. By six weeks post infection both the non-treated mice
and the mice treated with FK506 were producing less INF- than the previous sample,
however the treated mice were still producing more INF-. Mice treated with leptin were
producing about the same amount of INF- as mice treated with FK506. Mice treated
with CsA were producing the most INF- at six weeks post infection.
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44
were positive for the production of INF-; Figure 9 shows the percent of cells in the
sample that were positive for IL-4.
Three weeks post infection; animals treated with FK506 were producing less INF and IL-4 than infected non-treated animals. These animals were immunosuppressed.
By four weeks post infection, animals treated with FK506 were producing more INF-
and less IL-4 than infected non-treated animals, shifting the immune response toward a
TH1 type response. Six weeks post infection non-treated animals were producing the
least amount of INF- and IL-4. Mice treated with leptin were producing the most
amount of IL-4, indicating a strong TH2 type immune response. Mice treated with FK506
or CsA were producing similar amounts of INF-, however the mice treated with FK506
were producing more IL-4 than mice treated with CsA. The mice treated with FK506
never appeared to be ill; their feet did not swell or develop ulcers. The only sign of
illness was a swollen poplteal lymph node and a low titer of parasite from that same
lymph node.
FK506 was not found to be effective as a therapeutic drug with treatment until
27 days post infection (Figure 10). The footpads of the treated mice and the non-treated
mice began swelling at about the same time. The non-treated mice did experience
slightly more swelling than the treated mice.
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43
Figure 9: Amount of Intracellular IL-4 Produced
% Positive Cells
1.4
1.2
1
Non-treated
FK506
Leptin
CsA
0.8
0.6
0.4
0.2
0
Week 3
Week 4
Week 6
Weeks Post Infection
Figure 9: Amount of IL-4 Produced. Non-treated infected mice produced more IL-4
than mice treated with FK506 in weeks three and four post infection. Mice treated with
leptin produced a large amount of IL-4 at six weeks post infection. These mice were
very ill, with large foot pads, large ulcers, and large parasite loads.
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44
Figure 10: Average Footpad Thickness of Mice
Theraputically Treated with FK506
Average Footpad
Thickness (mm)
4
3.5
3
Controls
FK506
2.5
2
1.5
0
14
16
18
20
27
Days Post Infection
Figure 10: Average Footpad Thickness of Mice Therapeutically treated with FK506.
Treated and non-treated mice both developed footpad swelling at about the same time.
The treated mice displayed a smaller amount of swelling, but not by a large amount.
Shannon M. Potter
Chapter 6: Discussion
FK506 appears to be a very effective drug in preventing severe illness in BALB/c mice
due to Leishmania major infection. During both trials, mice treated with FK506 did not
show any signs of illness. The footpads of these mice only swelled a small amount. No
ulcers were observed during either trial. As shown in Figure 5, the amount of parasites
present in these mice was also much less than mice undergoing other treatment regimens.
These mice were shown to be immunosuppressed three weeks after infection, producing
very little of either cytokine (Figure 8 and Figure 9). Four weeks post infection these
mice were producing much more INF- than the non-treated mice and much less IL-4
than the non-treated mice (Figure 9). By six weeks post infection the mice treated with
FK506 had slowed down production of INF- while IL-4 production remained stable,
however, the amount of INF- was still greater than the amount of IL-4 being produced
(Figure 8 and Figure 9). This suggests that the suppression of the immune response early
in the disease process inhibited illness. It also suggests that the type of immune response
that was occurring later in the disease process was a TH1 type response.
Due to the success of the first trial, a therapeutic experiment was performed for
FK506. Results indicated that this particular treatment plan did not prevent disease
(Figure 10). Both sets of mice experience footpad swelling at about day 27 post
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46
infection. At this time, neither set of mice had developed ulcers. Based on the evidence
provided in the prophylatic intracellular cytokine data, I believe that if the theraputic
treatment had been started earlier this result may have been very different. I also believe
that the response would have been greater had we allowed the experiment to progress
longer.
The cyclosporin A group of mice suggested that CsA is effective in preventing the
mice from developing a severe reaction against the Leishmania infection. These mice
experienced weight loss during the treatment period, but readily gained weight after
treatment ended. CsA is a toxic drug that has caused death in previous treatment
regimens (unpublished data), however with the treatment regimen used in both trials, no
deaths occurred. Treated mice displayed some ruffling of fur during the treatment period,
indicating illness caused by toxicity (Table 1). In the second trial we again saw similar
toxicity data compared to the first trail. Although lower doses of cyclosporine may have
reduced toxicity, unpublished data suggests that they would be below therapeutically
beneficial levels.
These mice experienced some swelling of the footpad, which decreased with time
(Figure 5 and Figure 6). They also experienced high titers of parasites in their lymph
nodes, which also seemed to decrease as time passed (Figure 7). We observed cytokine
production only in week six, and again did not see a major difference between the CsA
treated animals and the infected, non-treated animals. Mice treated with CsA produced
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47
low levels of both cytokines; however, compared to the non-treated animals they were
producing more INF- (Figure 8). These mice produced about the same amount of IL-4
as the infected non-treated mice (Figure 9). The larger INF- production suggests that
treatment with CsA pushes the immune response toward a TH1 type response.
Mice in the leptin group fell somewhere between resistance and delay of illness
during the first trial. They did not experience any weight loss or ruffling of the fur (Table
1). However they did appear to have an increased amount of footpad swelling and ulcer
development (Figure 5). They also exhibited a large titer of parasites in their lymph nodes
but small titer in their spleens. In the second trial the dose of leptin was increased.
During the second trial the balance between TH1 and TH2 seemed to be shifted. Mice
developed ulcers and a large amount of footpad swelling before the infected non-treated
animals (Figure 6). Again, we observed cytokine production only in week six, and did not
see a significant difference between the leptin treated animals and the infected nontreated animals. Animals were producing more IL-4 than INF- six weeks post infection
(Figure 8 and Figure 9). The production of more IL-4 would indicate that leptin was
pushing the immune response towards a TH2 type response.
Based on the data presented, I believe that the immune response against
Leishmania major infection in BALB/c mice is responsible for either systemic disease or
resistance to the disease. Mice given FK506, which suppressed the immune system early
in the infection, never showed signs or symptoms of illness (Table 1, Figures 5, 6, 7, 8,
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48
and 9). The footpads of these mice did not swell or develop ulcers. Their fur remained
smooth, and their parasite load remained low. Data suggests that resistance to
leishmaniasis is determined early after infection. Future experiments should assay, cells
from each group of animals earlier in the disease process. I would also recommend that
therapeutic treatment using FK506 begin earlier after infection.
Shannon M. Potter
Chapter 7: References
1. Foote, Simon J., Hardman, Emanuel, and Roberts, Lynden J.(2000).
Leishmaniasis. BMJ, 321:801-804.
2. Behforouz, Nancy C., Mathison, Barry A., and Wenger, Charlotte D. Mathison,
Barry A. (1986). Prophylactic Treatment of BALB/c Mice with Cyclosporine A
and Its Analog B-5-49 Enhances Resistance to Leishmania major. J. of Immunol.
136:3067-3075.
3. Roberts, Larry S., and Schmidt, Gerald D. (2000). Foundations of Parasitology.
Fourth Edition
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